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Journal of Bacteriology, October 2000, p. 5765-5770, Vol. 182, No. 20
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

A Truncated Soluble Bacillus Signal Peptidase Produced in Escherichia coli Is Subject to Self-Cleavage at Its Active Site

M. L. van Roosmalen, J. D. H. Jongbloed, A. Kuipers, G. Venema, S. Bron, and J. M. van Dijl*

Department of Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, 9750 AA Haren, The Netherlands

Received 1 May 2000/Accepted 19 July 2000

Soluble forms of Bacillus signal peptidases which lack their unique amino-terminal membrane anchor are prone to degradation, which precludes their high-level production in the cytoplasm of Escherichia coli. Here, we show that the degradation of soluble forms of the Bacillus signal peptidase SipS is largely due to self-cleavage. First, catalytically inactive soluble forms of this signal peptidase were not prone to degradation; in fact, these mutant proteins were produced at very high levels in E. coli. Second, the purified active soluble form of SipS displayed self-cleavage in vitro. Third, as determined by N-terminal sequencing, at least one of the sites of self-cleavage (between Ser15 and Met16 of the truncated enzyme) strongly resembles a typical signal peptidase cleavage site. Self-cleavage at the latter position results in complete inactivation of the enzyme, as Ser15 forms a catalytic dyad with Lys55. Ironically, self-cleavage between Ser15 and Met16 cannot be prevented by mutagenesis of Gly13 and Ser15, which conform to the -1, -3 rule for signal peptidase recognition, because these residues are critical for signal peptidase activity.

* Corresponding author. Present address: Department of Pharmaceutical Biology, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands. Phone: 31503633079. Fax: 31503632348. E-mail: J.M.VAN.DIJL{at}FARM.RUG.NL.

Journal of Bacteriology, October 2000, p. 5765-5770, Vol. 182, No. 20
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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